Hierarchy‐Assembled Dual Probiotics System Ameliorates Cholestatic Drug‐Induced Liver Injury via Gut‐Liver Axis Modulation

Abstract Cholestatic drug‐induced liver injury (DILI) induced by drugs or other xenobiotics is a severe and even fatal clinical syndrome. Here, living materials of hierarchy‐assembled dual probiotics system are fabricated by sequentially encapsulating probiotic Lactobacillus delbrueckii subsp. bulgaricus (LDB) and Lactobacillus rhamnosus GG (LGG) into Ca2+‐complexed polymer microspheres for effective prevention of cholestatic DILI. Upon entering intestinal tract of the constructed living materials, LGG is released because of pH‐triggered dissolution of outer enteric polymer coating. The released LGG can inhibit hepatic bile acids (BAs) synthesis by activating intestinal farnesoid X receptor‐fibroblast growth factor 15（FGF‐15) signaling pathway. BAs excretion is also facilitated by LGG through increasing the abundance of bile salt hydrolase (BSH)‐active gut commensal bacteria. Furthermore, exposed positively‐charged chitosan shell can absorb the excessive BAs via electrostatic interaction, which leads to steady BAs fixation by the imprisoned LDB, decreasing the total BAs amounts in enterohepatic circulation. Together, the fabricated living materials, obtained here, can effectively prevent cholestatic DILI through dredging cholestasis via gut‐liver axis modulation. The therapeutic effect is demonstrated in α‐naphthylisothiocyanate and clinical antiepileptic drug valproate acid‐induced cholestatic DILI mouse models, which reveal the great potential for effective cholestatic DILI management.


Instrumentations.
Scanning electron microscope (SEM) image was obtained by using scanning electron microscope (Zeiss Sigma). Blood biochemistry analysis was performed by biochemical auto analyzer (MNCHIP, Tianjin, China). In vivo imaging was carried out by Spectrum Pre-clinical In Vivo Imaging System (PekinEmer). Fluorescent images were obtained by fluorescence inverted microscope (Olympus IX73P2F). The UV-vis absorbance was measured by UV-vis spectroscopy (Lambda Bio40). The bacterial density was measured using microplate reader (Bio-Rad, Model 550, USA). The zeta potential was measured by a zeta sizer (Nano ZS, Malvern Instruments).
Fluorescent labeling of LDB, LGG and chitosan. 8 mL LDB in MRS broth culture medium S3 was centrifuged (6000 rpm, 3 min), followed by resuspending in 8 mL PBS. Then, 7-Hydroxycoumarin-3-carboxylic acid N-succinimidyl ester (5 mg/mL, 10 μL, dissolved in DMSO) was added to above solution. After an hour of stirring in dark at room temperature, the reactant was centrifuged, and precipitate was washed with PBS until no fluorescence in supernatant.
1 g chitosan was dissolved in 100 mL of 1% acetic acid solution (w/v), then 100 μL of FITC (0.1 mg/mL, dissolved in DMSO) was added. After an hour of stirring in dark at room temperature, the reactant was dialyzed, follow by freeze drying. The lyophilized powders of FITC-labeled chitosan were redispersed in 100 mL of 1% acetic acid solution (w/v).
Synthesis of hierarchy-assembled dual probiotics system. The synthesis of hierarchyassembled dual probiotic system (LCA/LGG-LDB) was divided into three steps. Firstly, LDB-encapsulated calcium alginate microspheres were prepared by microemulsion method.
Briefly, CaCl 2 solution (0.5 M, 5 mL) was added to 40 mL soybean oil dropwise under vigorous stirring at room temperature. LDB solution (OD 600 = 1.5, 5 mL) was mixed with 5 mL of 1% sodium alginate (w/v), and then was added into 40 mL soybean oil drop by drop under vigorous stirring. Then, sodium alginate-contained soybean oil was added to the CaCl 2contained soybean oil quickly. After one hour of reaction at room temperature, a few drops of Tween 80 were added, followed by centrifugation (3000 rpm, 2 min). The supernatant was discarded and these microspheres were washed by CaCl 2 solution.
Next, the resulting microspheres were dispersed in 10 mL of 1% chitosan solution (w/v), followed by one hour of stirring at room temperature. Then, the chitosan-modified microspheres were washed by phosphate-buffered saline (PBS) twice, and then were crosslinked with genipin (10 mM, 10 mL) for 2 h.
Lastly, these microspheres were dispersed into 10 mL CaCl 2 (0.5 M) solution, followed by mixing with LGG solution (OD 600 = 1.0, 5 mL). Injection pump was used to inject 10 mL S4 L100-55 solution (8 mg/mL) into the mixture at the flow rate of 20 mL/h for 30 min under gentle stirring condition. Then, HCl solution (pH = 1) was added to the mixture to maintain pH = 4.5. After centrifugation (800 rpm, 1 min), microspheres were collected, and then stored in 12.5 mL HCl solution (pH = 4.5) at 4 o C for use. LCA/LDB, LCA/LGG and LCA were prepared in the same way.
Survival of bacteria in SGF, SIF and BA. 1 mL LCA/LGG solution was added to 9 mL PBS (pH = 7.4), SGF (pH = 3.0, 3.2 g/L pepsin and 2 g/L NaCl), SIF (pH = 6.8, 10 g/L trypsin and 6.8 g/L KH 2 PO 4 ), and sodium cholate (0.3 mg/mL), respectively. These mixtures were incubated with a gentle stirring for 100 rpm at 37 o C for 1.5 h. After a 3-minute-centrifugation, precipitate was washed with PBS, and then resuspended in 5 mL PBS. After proper dilution, 100 μL solution was coated onto MRS agar plates. The bacterial colonies were counted after a 24-hour-incubation anaerobically at 37 o C.
Adsorption tests of sodium cholate. 1.2 mL of the synthesized CSA-LDB, CSA microspheres or LDB (OD 600 = 1.5) stored in 5 mL PBS were mixed with 12 mL sodium cholate solution (3 mg/L), then were stirred gently at 37 o C. After centrifugation, the supernatant was collected and filtered with a 0.22-μm membrane. Then, 1 mL of the filtered supernatant was added into a 12 mL glass tube and 6 mL of 64% H 2 SO 4 was subsequently added. After fully mixing, 1 mL of 0.3% furfural was added and reacted for 30 min at 65 o C.
After cooling to room temperature, the absorbance at 620 nm were measured. In this study,

In vivo gastrointestinal retention evaluation. In vivo gastrointestinal retention of LCA/LDB
were evaluated with healthy female C57BL/6 mice. Mice were fasted for 12 h before orally administrating Cy5.5-labeled LCA/LDB (300 μL). Then, mice were imaged using IVIS at predetermined time points (0 h, 3 h, 6 h, 9 h and 12 h after administration). LCA/LDB without chitosan modification was used as control group. After IVIS imaging, mice were euthanized, and gastrointestinal tracts were collected and imaged.
ANIT-induced cholestasis and liver injury model. C57BL/6 female mice were randomly divided into six groups: control group, ANIT group, ANIT + LCA group, ANIT + LCA/LDB group, ANIT + LCA/LGG group and ANIT + LCA/LGG-LDB group. Mice in latter five groups were orally administrated with 100 μL of ANIT (12 mg/mL, dissolved in soybean oil, once a day for two days), while mice in control group were administrated with equal volume of soybean oil. Two days later, mice in latter five groups were gavaged with 500 μL of the stored materials once a day for twelve days, while mice in control group were administrated with equal volume of PBS. After a-twelve-days treatment, faeces were collected for further analysis. Then, all mice were euthanized, peripheric blood, liver tissue and ileum were collected for further tests and analysis.

VPA-induced cholestasis and liver injury model. In VPA-induced cholestasis and liver
injury model, mice were randomly divided into three groups: control group, VPA-treated group (VPA), as well as LCA/LGG-LDB treated group (VPA+LCA/LGG-LDB). Mice were administrated with 100 μL of VPA (100 mg/mL) via oral administration in every morning for two weeks, while the control group were orally administrated with equal volume of PBS. In the tenth day, mice in treatment group were orally gavaged with 500 μL of LCA/LGG-LDB in every night for twenty days. Mice in control group and VPA group were administrated with equal volume of PBS. In the end of this experiment, all mice were sacrificed, and then blood, faeces, liver tissue and ileum were collected for further tests and analysis.
S6 Serum biochemical analysis. Serum was collected by centrifugation from whole blood sample at the speed of 3200 rpm at 4 o C for 15 min. Serum ALP, ASP, AST, total bilirubin and direct bilirubin were detected by liver and kidney function test lyophilization kit (MNCHIP) according to the manufacturer's protocol.
Determination of BSH in mouse faeces. Briefly, 0.5 mL of lysozyme solution (0.5 mg/mL) was added to a mouse fecal sample, and then mixture was incubated at 37 o C for 10 minutes.
After homogenization, the mixture was centrifuged at 4 o C, and supernatant was collected. Statistical Analysis. All values (n ≥ 3) were represented as the mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism 8.0 software and two-tailed unpaired Student's t-test or one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test was used to compare differences between treatment groups.
The degradation behavior of LCA/LGG-LDB in (A) SGF (pH = 3, 1.5 h) and (B) SIF (pH = 6.8, 1.5 h). The enlarged image showed the released LGG in SIF while it was absent in SGF.
Scale bar: 50 μm. S12 Figure S6. The images of the excreted microspheres in faeces (Scale bar: 100 μm). Direct bilirubin. The data were presented as the mean ± s.d., n = 3. The statistical significance was calculated via one-way ANOVA with Tukey's multiple comparisons test. *P < 0.05.